The aflatoxins are a paradigm among mycotoxins and occupy a central place in environmental toxicology. Hepatocarcinomas are the third most common cause of cancer death in the world. Hepatitis infections and dietary aflatoxin are major risk factors contributing to the incidence of this disease. The aflatoxigenic Aspergillus species, A. parasticus, A. flavus and A. nomius, commonly infect grains and food stuffs where clear epidemiological data correlate to human disease. Chronic ingestion is a major cause of premature death in Asia, Africa and Central America. A direct link has been forged between the interaction of the metabolically activated form of the toxin and DNA, particularly in a hot spot in the p53 gene leading to mutation of its important encoded cell cycle regulating protein. Understanding its biosynthesis will lead to control of this environmental carcinogen. The aflatoxins are created by an unusually long and complex biosynthesis. Key molecular rearrangements, some remarkable mechanistically, are catalyzed by cytochromes P450. The mechanisms of these cleavage and rearrangement reactions will be studied and modeled by chemical mimetics to understand their underlying chemistry. The second, and principal, goal is to capitalize upon exciting progress made in the current grant period to understand the function of non-reducing iterative polyketide synthases central to aflatoxin biosynthesis and many other fungal natural products. Of the three principal types of PKSs, least is known about the programming of iterative Type I systems;that is, how are starter units recruited and synthesis begun, how is chain length determined, how is the canonical and intrinsically reactive poly 2-keto intermediate stabilized, how is redox state controlled during chain elongation, and, finally, how are intramolecular cyclizations controlled to specific ring forms in preference to others. Superimposed on this is the fundamental question of iterative catalysis, a rare but impressively efficient process in which active sites in these polydomainal enzymes are used over and over again, accommodating a sequence of growing substrates yet faithfully executing a synthetic program. We have discovered two previously unrecognized domains in these domains we now believe are general to this class of enzymes, a starter unit:acyl-carrier protein transacylase (SAT) domain, which might account for the classical observation of a starter unit effect, and a product template (PT) domain, which in preliminary results clearly has a role in polyketide stabilization and product templating. Biochemical, X-ray crystallographic and mass spectrometric experiments are outlined to understand the functions of these domains individually and working together in what is already emerging as a new picture of iterative catalysis. PUBLIC HEALTH RELEVANCE The aflatoxins occupy a central place in environmental toxicology. Hepatitis infections and dietary aflatoxin from infected grains and foodstuffs are major risk factors for hepatocarcinoma, the third most common cause of cancer death in the world. Understanding its biosynthesis will lead to its control.